Estrogen-based drugs are used widely for treatment areas ranging from breast cancer to menopause to contraception, but all of them suffer limitations due to unwanted effects of the drugs in other tissues. The long-term goal of this proposed research is to elucidate the molecular details of estrogen signaling in the context of other signaling pathways in the cell and how crosstalk with these signaling pathways dictate the response profiles of estrogen-mimicking drugs. Growing evidence suggests that steroid hormones can elicit responses from receptors located outside of the nucleus. Possible receptors for these responses, which have been noted for all steroid hormones, range from novel classes of membrane-spanning receptors to G-protein coupled receptors located on endoplasmic reticulum to different forms of the classic steroid hormone receptor. We hypothesize that targeting estrogen ligands to certain parts of the cell could selectively modulate some of these responses. We propose to carry out several different studies of different types of estrogen-regulated responses focusing on how ligand structure and receptor localization play a role in modulating different types of responses to estrogenic and antiestrogenic compounds. Aim 1 is focused on the synthesis of novel macromolecular conjugates of different estrogen-mimicking analogs that localize to different parts of the cell. We have found that 4-hydroxytamoxifen conjugated to different scaffolds can localize to the nucleus, cytoplasm, or outer plasma membrane. Whether other estrogen ligands can elicit the same pattern of localization will be explored. Aim 2 is focused on investigating how ligand structure and localization affects a number of different estrogen responses. Our previous results support the hypothesis that the structure-activity relationships underlying many of these crosstalk-dependent responses are significantly different than those underlying classic estrogen receptor activity, and Aim 2 will focus on assays looking at a number of diverse responses to estrogen and whether localization of the ligand to certain parts of the cell can also play a role. In aim 3, we propose to investigate whether the properties of the conjugates themselves are responsible for some of their biological effects and explore possible effects of multivalency and conjugation chemistry. Aim 4 will look at the functional actions of the conjugates on the different estrogen receptors and on a number of cellular responses to estrogenic and antiestrogenic compounds.